Asymmetrical distribution of the transcriptionally competent NORs in mitosis

Markéta Kalmárová,Sánchez Pilar Testillano,Evgeny Smirnov,Alexey Popov,Lubomír Kováčik

doi:10.1016/j.jsb.2008.04.002

Abstract

Ribosomal genes are organized in clusters termed Nucleolus Organizer Regions (NORs). Essential components of the RNA polymerase I transcription machinery, including Upstream Binding Factor (UBF), can be detected on some NORs during mitosis; these NORs, termed competent, are believed to be transcriptionally active during interphase. In cultured mammalian cycling cells, the number of competent NORs, and their distribution among the different chromosomes, does not vary significantly in the sequential cell cycles. In this work we investigate whether this stable state is achieved by equal distribution of competent NORs during cell division. To answer this question we first studied the state of NORs in telophase HeLa and LEP cells. In both cell lines we found a small but significant difference between the emerging daughter cells in the number of UBF-loaded NORs. To reveal the cause of this difference, we followed the fate of individual NOR using HeLa derived cell line stably expressing UBF-GFP. We demonstrated that some NORs in metaphase are “asymmetrical”, i.e. they lack the signal of competence on one of the sister chromatids. Regular presence of such NORs can account for the difference in the number of competent NORs obtained by the daughter cells emerging in mitosis.

Highlights

Ribosomal genes coding 5.8S, 18S and 28S rRNA are organized in clusters which can be identified in mitotic chromosomes and are called Nucleolus Organizer Regions (NORs)
Following the segregation of competent NORs during mitosis in vivo in a HeLa derived cell line stably expressing Upstream Binding Factor (UBF)-GFP (HeLa-UBF), we found that chromosomes with asymmetrical NORs, in which only one of sister chromatids carries the signal of competence, are the main source of the observed mitotic asymmetry
We observed on mitotic chromosomes 6-12 competent NORs in HeLa and 6-10 competent NORs in LEP cells, using antibodies against human UBF, mouse UBF and polymerase I (pol I)

Summary

Introduction

Ribosomal genes coding 5.8S, 18S and 28S rRNA are organized in clusters which can be identified in mitotic chromosomes and are called Nucleolus Organizer Regions (NORs). RDNA transcription is efficiently blocked from prophase to late anaphase, at least some subunits of RNA polymerase I (pol I) along with its main transcription factors, the Upstream Binding Factor (UBF) and promoter selectivity complex (SL1), can be detected on certain NORs even in metaphase (Babu and Verma, 1985; Weisenberger and Scheer, 1995; Jordan et al, 1996; Roussel et al, 1996; Gebrane-Younes et al, 1997; Sirri et al., 1999; Leung et al, 2004; Prieto and McStay, 2005) Such NORs, termed “transcriptionally competent” or “competent” (Dousset et al, 2000; Savino et al, 2001), can be visualized on the chromosome spreads by silver staining (Howell and Black, 1980). UBF, the most abundant component of the competent NORs, serves apparently as an architectural element responsible for transcriptionally favorable state of chromatin (O’Sullivan et al, 2002; Mais et al, 2005; Prieto and McStay, 2007)

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